RESUMO
Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling.
Assuntos
NF-kappa B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Regiões 3' não Traduzidas/genética , Tirosina Quinase da Agamaglobulinemia , Pareamento de Bases/genética , Núcleo Celular/metabolismo , Citocinas/biossíntese , Regulação para Baixo/genética , Humanos , Fosforilação , Regiões Promotoras Genéticas/genética , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Lentiviral vectors deliver antigens to dendritic cells (DCs) in vivo, but they do not trigger DC maturation. We therefore expressed a viral protein that constitutively activates NF-kappaB, vFLIP from Kaposi's sarcoma-associated herpesvirus (KSHV), in a lentivector to mature DCs. vFLIP activated NF-kappaB in mouse bone marrow-derived DCs in vitro and matured these DCs to a similar extent as lipopolysaccharide; costimulatory markers CD80, CD86, CD40, and ICAM-1 were upregulated and tumor necrosis factor alpha and interleukin-12 secreted. The vFLIP-expressing lentivector also matured DCs in vivo. When we coexpressed vFLIP in a lentivector with ovalbumin (Ova), we found an increased immune response to Ova; up to 10 times more Ova-specific CD8(+) T cells secreting gamma interferon were detected in the spleens of vFLIP_Ova-immunized mice than in the spleens of mice immunized with GFP_Ova. Furthermore, this increased CD8(+) T-cell response correlated with improved tumor-free survival in a tumor therapy model. A single immunization with vFLIP_Ova also reduced the parasite load when mice were challenged with OVA-Leishmania donovani. In conclusion, vFLIP from KSHV is a DC activator, maturing DCs in vitro and in vivo. This demonstrates that NF-kappaB activation is sufficient to induce many aspects of DC maturation and that expression of a constitutive NF-kappaB activator can improve the efficacy of a vaccine vector.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/virologia , Lentivirus/genética , NF-kappa B/biossíntese , Proteínas Virais/imunologia , Vacinas Virais/genética , Animais , Vacinas Anticâncer/imunologia , Citocinas/biossíntese , Leishmania donovani/imunologia , Leishmaniose/prevenção & controle , Lentivirus/imunologia , Camundongos , Neoplasias/imunologia , Ovalbumina/imunologia , Receptores Imunológicos/biossíntese , Baço/imunologia , Análise de Sobrevida , Proteínas Virais/genética , Vacinas Virais/imunologiaRESUMO
In the present report we have determined the molecular mechanisms, which govern the expression of the human IL-10 gene when induced by the glucocorticoid Methyl-Prednisolone (MP). Treatment of cells with MP at 10(-6) M will readily induce IL-10 in CD19+ primary B cells and in a human B cell line. Analysis of the IL-10 promoter showed a robust 18-fold induction and demonstrated that a potential GRE motif was not required, while mutation of the -120 STAT-motif strongly reduced MP-induced trans-activation. A strong induction was also seen with a trimeric STAT-motif and over-expression of dominant-negative STAT3 could block MP induction of IL-10 mRNA. Finally, MP treatment induced binding of STAT3 to the promoter as shown by gelshift, supershift and by chromatin-immunoprecipitation. These data show that glucocorticoid-induced expression of the IL-10 gene is mediated by the transcription factor STAT3.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-10/genética , Metilprednisolona/farmacologia , Fator de Transcrição STAT3/metabolismo , Adenoviridae , Linhagem Celular , Imunoprecipitação da Cromatina , Genes Dominantes , Genes Reporter , Humanos , Interleucina-10/metabolismo , Luciferases/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de SequênciaRESUMO
Tumor-derived microvesicles (TMV) can mimic effects of tumor cells leading to an increased anti-inflammatory cytokine production, such as interleukin 10 (IL-10), by tumor-infiltrating monocytes and macrophages. Yet, the mechanism of IL-10 induction by TMV in monocytes remains unclear. The co-incubation of TMV derived from the human pancreas carcinoma cell line (HPC-4) with human monocytes resulted in a nearly 30-fold increase in IL-10 protein production. This effect operates at the level of transcription since monocytes transduced with an adenovirus containing IL-10-promoter luciferase reporter gene showed a 5-fold induction of luciferase activity after treatment with TMV. Since tumor cells can express hyaluronan (HA), which participates in tumor invasion and metastases, we have tested its effect on IL-10 expression. We showed that HA at the concentration of 100µg/ml induces IL-10 protein expression and the IL-10 promoter activation in monocytes. Moreover, hyaluronidase treatment of TMV reduced IL-10 protein production by 50% and promoter activity by 40%. Inhibitors of the PI3K/Akt/mTOR pathway reduced both, TMV-induced IL-10 promoter activity and protein production, and the same was observed in monocytes when stimulated by HPC-4 cells or HA. Inhibition of PI3K activity down-regulated phosphorylation of the Akt and (to a lesser extent) mTOR proteins in monocytes following TMV or HA stimulation. When comparing monocyte subsets, TMV induced IL-10 protein and mRNA synthesis only in classical CD14++CD16- but not in CD16-positive monocytes. Our data show that TMV induce IL-10 synthesis in human classical monocytes via HA, which, in turn, activates the PI3K/Akt/mTOR pathway.
Assuntos
Micropartículas Derivadas de Células , Ácido Hialurônico/administração & dosagem , Interleucina-10/biossíntese , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Modelos Biológicos , Monócitos/imunologia , Neoplasias/metabolismo , Receptores de IgG/metabolismoRESUMO
IL-10 is an important immunosuppressive cytokine that can down-regulate expression of other cytokines and has been shown to down-regulate itself. We show, in this study, that treatment of human monocyte-derived macrophages with IL-10 induces IL-10 mRNA in a dose- and time-dependent manner with an optimum induction at 100 ng/ml and at 6 h, whereas IL-10-induced IL-10 protein can be detected at 18 h. In the same cells, IL-10 can partially suppress IL-10 mRNA induced by LPS, but only down to the level of IL-10-induced IL-10. An adenoviral luciferase reporter construct driven by the -195 IL-10 promoter, which contains a Stat motif, was readily induced by both IL-10 and LPS. Mutation of this Stat motif ablated IL-10 activation of this promoter, but not the LPS activation. Finally, we show that overexpression of a dominant-negative Stat3 protein will prevent IL-10 induction, but not LPS induction, of IL-10 mRNA. These data show that IL-10 induces IL-10 in monocyte-derived macrophages in an autocrine manner via activation of the transcription factor Stat3.
Assuntos
Interleucina-10/farmacologia , Macrófagos/imunologia , Monócitos/citologia , Fator de Transcrição STAT3/fisiologia , Adenoviridae/genética , Humanos , Interleucina-10/genética , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/análiseRESUMO
Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.
Assuntos
Proteínas de Bactérias/genética , Etnicidade , Sistema Imunitário/fisiologia , Mutação , Mycobacterium tuberculosis , Ásia , Proteínas de Bactérias/metabolismo , Células Cultivadas , Citocinas/imunologia , Transmissão de Doença Infecciosa , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Monócitos/citologia , Monócitos/metabolismo , Monócitos/microbiologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Fagócitos/citologia , Fagócitos/metabolismo , Fagócitos/microbiologia , Fenótipo , Polimorfismo Genético , Tuberculose/epidemiologia , Tuberculose/imunologia , Tuberculose/transmissão , Reino Unido/epidemiologiaRESUMO
Efforts to identify the signal transduction pathways used by interleukin-10 (IL-10) have resulted in limited success. The anti-inflammatory effects elicited by IL-10, and the mechanisms by which these are mediated, are still relatively unknown. Understanding the signalling mechanisms behind the suppression of cytokine expression by IL-10 could be of potential therapeutic interest. Although the consensus is that the Janus kinase, Jak1, as well as the signal transducer and activator of transcription STAT3 are central, much controversy exists about the participation and roles of many other signalling pathways targeted by IL-10. The mechanisms of cytokine suppression proposed by various groups have included transcriptional, post-transcriptional and post-translational regulation of IL-10 target genes; nevertheless no unifying model has emerged thus far. Here we would like to highlight novel findings and discuss their implications in the context of current understanding of IL-10 signalling.